银杏蜜环口服溶液作用p38 MAPK调节eNOS/NO信号途径保护人脐静脉内皮细胞缺氧复氧损伤  

Yinxing Mihuan Oral Solution Protect HUVECs Against Hypoxia Reoxygenation Injury Through p38 MAPK Regulating e NOS/NO Signaling Pathway

作　　者：韩笑[1] 徐立[1] 刘建勋[1] 王益民 王勇 

机构地区：[1]中国中医科学院西苑医院基础医学研究所北京市中药药理重点实验室,北京100091 [2]西安步长中医心脑病医院,西安710082

出　　处：《中国实验方剂学杂志》2017年第23期79-84,共6页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家重点基础研究发展计划(973计划)项目(2015CB554405);中国中医科学院科技创新团队项目(YS1303)

摘　　要：目的:评估银杏蜜环口服溶液对缺氧复氧损伤人脐静脉内皮细胞(HUVECs)的保护作用;从p38丝裂原活化蛋白激酶(p38 MAPK)及内皮型一氧化氮合酶(e NOS)/一氧化氮(NO)信号途径探讨银杏蜜环口服溶液保护受损HUVECs的药效机制。方法:缺氧缺糖/复氧复糖法建立体外HUVECs缺血再灌注损伤模型。分为正常组、模型组、银杏蜜环口服溶液高质量浓度组(75 mg·L-1,简称银蜜高),低质量浓度组(36 mg·L-1,简称银蜜低),p38 MAPK抑制剂SB203580组,银蜜高+SB203580组,e NOS抑制剂亚硝基左旋精氨酸甲酯(L-NAME)组,银蜜高+L-NAME组。细胞增殖-毒性检测试剂盒(CCK-8)及微量酶标法检测细胞活力及细胞损伤;蛋白免疫印迹法(Western blot)检测磷酸化p38 MAPK(p-p38 MAPK)/p38 MAPK和磷酸化e NOS(p-e NOS)/e NOS蛋白表达;酶联免疫吸附法(ELISA)检测肿瘤坏死因子-α(TNF-α),可溶性细胞间黏附分子-1(s ICAM-1),半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)含量;硝酸盐还原酶法检测NO含量。结果:银蜜高、银蜜低组可显著提高缺氧复氧损伤HUVECs活力、降低细胞乳酸脱氢酶(LDH)漏出量(P<0.05,P<0.01);抑制受损细胞p38的磷酸化(P<0.05),降低细胞培养上清中TNF-α,s ICAM-1含量(P<0.05,P<0.01)。在给予银蜜高或(和)SB203580后,受抑制的e NOS磷酸化表达水平显著升高(P<0.05);银蜜高可显著提高受损细胞NO含量,在加入e NOS抑制剂L-NAME后,银蜜高可拮抗L-NAME降低细胞NO生成和升高Caspase-3的作用(P<0.05)。结论:银杏蜜环口服溶液可提高氧复氧损伤HUVEs活力,具有抗炎、调节内皮系统、抑制细胞凋亡的作用,机制与其作用于p38 MAPK进而调节e NOS-NO信号途径相关。Objective： To study the effect and mechanism of Yinxing Mihuan oral solution on human umbilical vein endothelial cells （HUVECs） injured by hypoxia/ reoxygenation （H/R） through p38 mitogen activated protein kinase （p38 MAPK） regulating endothelial nitric oxide synthase （eNOS） /nitric oxide （NO）signaling pathway. Method： Hypoxia ischemia/reperfusion injury model was established by hypoxia/hypoglycemia/ reoxygenation on HUVECs. Cultured HUVECs were divided into control group, H/R group, Yinxing Mihuan oral solution high （75 mg·L^-1） , low dose group （36 mg·L^-1） , p38 MAPK inhibitor SB203580, Yinxing Mihuan oral solution high dose ＋ SB203580, eNOS inhibitor L-nitro-arginine methylester （L-NAME）, Yinxing Mihuan oral solution high dose ＋ L-NAME. Cell viability and cell injury were measured by cell counting kit-8 （CCK-8） and enzyme labeling method. Protein expression of phosphorylating-p38 MAPK （p-p38 MAPK） /p38 MAPK and phosphorylating-eNOS （p-eNOS） / eNOS were detected by Western blot. The levels of tumor necrosis factor-α（TNF-α）, soluble intercellular adhesion molecule-1 （sICAM-1）, cysteine aspartate protease-3 （Caspase-3） were determined by enzyme-linked immunosorbent assay （ELISA）. NO content was measured by nitrate reductase method. Result： Yinxing Mihuan oral solution high and low dose group could significantly improve cell viability and reduce lactate dehydrogenase （LDH） content （P 〈 0.05, P 〈 0.01 ） , inhibit p-p38 MAPK expression on injured HUVECs, decrease TNF-α, sICAM-1 levels in cell culture supernatant （P 〈 0.05, P 〈 0.01 ）. The inhibited p-eNOS expression was significantly increased after administration of Yinxing Mihuan oral solution high dose and （or） SB203580 （P 〈 0.05）. Yinxing Mihuan oral solution high dose significantly increased the NO content of damaged HUVECs. After the addition of the eNOS inhibitor L-NAME, Yinxing Mihuan oral solution high dose could antagonize the effects of L-NAME on decr

关 键 词：银杏蜜环口服溶液 缺氧复氧 p38丝裂原活化蛋白激酶(p38 MAPK) 内皮型一氧化氮合酶(e NOS) 一氧化氮(NO) 

分 类 号：R285.5[医药卫生—中药学]